ABSTRACT We have identified a subset of ciliated cells (30%) in the mouse and human airways that express MIWI2, a protein only previously observed in the mouse testes where it functions to suppress transposon transcription. Newly generated ontogeny data along with global transcriptomic and gene ontology analyses of mouse MIWI2-positive (MPACs) and negative ciliated cells (non-MPACs) further support the concept of multiciliated cell heterogeneity and our overall hypothesis: MIWI2-positive expression distinguishes 2 distinct ciliated cell lineages in the airway. For the first time, these data hint at functionally distinct multi-ciliated cell subsets and suggest new lineage models for the airway epithelium. In view of the broader implications of this work for the field, our over-riding objective and focus in this revised grant are to develop and elucidate a basic understanding of the differential biology of these 2 subtypes of ciliated cells in the mouse airway. As a result, many of our studies are now deliberately and logically directed at characterizing the ontogeny, half-life, and origins of MPACs relative to non-MPACs. Our data also indicate that ciliated cells play a distinct role in regulating inflammation during injury, a function not generally ascribed to this cell type. Determining the relative role of MPACs and non-MPACs and ciliated MIWI2 in regulating inflammatory states in the lung are thus important sub-goals of this proposal. In our view, identifying immune-regulatory roles for ciliated cells has not been sufficiently addressed since most of the research on this cell type has traditionally concentrated on their mechanical contributions to mucus clearance. As such, this direction lends a deeper and more expansive significance to our proposed studies. By using several unique genetic mouse models that sustain the identification, tracking, and fate of ciliated cells, including MPACs, we propose to examine our hypothesis and meet our goals in 3 Aims. In Aim 1 we well establish the ontogeny of MPACs, their lineage relationship to non-MPACS and their half-life during lung homeostasis. In Aim 2, we will identify the origin of new MPACs that arise during airway remodeling after lung infection and during MPAC- specific cell regeneration. In Aim 3, we will determine the relative roles of MIWI2 protein and MPACs in the host response to early lung inflammation and infection. Together, these studies will forge new directions in the fundamental biology of the airway by establishing a new understanding of epithelial cell lineages and by integrating and connecting the fields of Piwi proteins, inflammation, and multiciliated cells of the lung.